THE JOURNAL OF BIOLOGICAL CHEMISTRY
© 2002 by The American Society for Biochemistry and Molecular Biology, Inc.
Vol. 277, No. 6, Issue of February 8, pp. 3836 –3841, 2002
Printed in U.S.A.
Structural Features of tRNALys Favored by Anticodon Nuclease as
Inferred from Reactivities of Anticodon Stem and Loop Substrate
Analogs*
Received for publication, October 18, 2001, and in revised form, November 20, 2001
Published, JBC Papers in Press, November 26, 2001, DOI 10.1074/jbc.M110072200
Yue Jiang‡§, Shani Blanga‡, Michal Amitsur‡, Roberto Meidler‡, Eli Krivosheyev‡,
Mallikarjun Sundaram¶, Ashok C. Bajji¶, Darrell R. Davis¶, and Gabriel Kaufmann‡储
From the ‡Department of Biochemistry, Tel Aviv University, Ramat, Aviv 69978, Israel and the ¶Department of Medicinal
Chemistry, University of Utah, Salt Lake City, Utah 84112
The bacterial tRNALys-specific PrrC-anticodon nuclease efficiently cleaved an anticodon stem-loop (ASL) oligoribonucleotide containing the natural modified bases,
suggesting this region harbors the specificity determinants. Assays of ASL analogs indicated that the 6-threonylcarbamoyl adenosine modification (t6A37) enhances
the reactivity. The side chain of the modified wobble
base 5-methylaminomethyl-2-thiouridine (mnm5s2U34)
has a weaker positive effect depending on the context of
other modifications. The s2U34 modification apparently
has none and the pseudouridine (⌿39) was inhibitory in
most modification contexts. GC-rich but not IC-rich
stems abolished the activity. Correlating the reported
structural effects of the base modifications with their
effects on anticodon nuclease activity suggests preference for substrates where the anticodon nucleotides assume a stacked A-RNA conformation and base pairing
interactions in the stem are destabilized. Moreover, the
proposal that PrrC residue Asp287 contacts mnm5s2U34
was reinforced by the observations that the mammalian
tRNALys-3 wobble base 5-methoxycarbonyl methyl-2thiouridine (mcm5s2U) is inhibitory and that the D287H
mutant favors tRNALys-3 over Escherichia coli tRNALys.
The detection of this mutation and ability of PrrC to
cleave the isolated ASL suggest that anticodon nuclease
may be used to cleave tRNALys-3 primer molecules annealed to the genomic RNA template of the human immunodeficiency virus.
A tRNALys-specific anticodon nuclease (ACNase)1 in latent
form is encoded by the optional Escherichia coli prr locus (1). It
comprises the core ACNase polypeptide PrrC and type IC DNA
restriction-modification enzyme EcoprrI that masks PrrCs activity (2–5). The phage T4-coded peptide Stp inhibits EcoprrI
DNA restriction and activates ACNase (6). Cleavage of
* This work was supported in part by grants from the Israeli National
Science Foundation, the Israeli Ministry of Science Canadian-Israeli
Cooperation Fund, and work at the University of Utah was supported
by National Institutes of Health Grant GM55508. The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked “advertisement”
in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ Supported in part by postdoctoral fellowship of the Israeli Council
for Higher Education.
储 Incumbent of the Louise and Nahum Barag Chair in Cancer Molecular Genetics. To whom all correspondence should be addressed. Tel.:
972-3-640-9067; Fax: 972-3-640-6834; E-mail: gabika@tauex.tau.ac.il.
1
The abbreviations used are: ACNase, anticodon nuclease; ASL, anticodon stem and loop; t6A, threonylcarbamoyl adenosine; mnm5s2U,
5-methylaminomethyl-2-thiouridine; mcm5s2U, 5-methoxycarbonyl
methyl-2-thiouridine.
tRNALys 5⬘ to the wobble base ensues, yielding 2⬘:3⬘-cyclic
phosphate (2⬘:3⬘-p⬎) and 5⬘-OH termini. However, the lesion is
normally offset by the phage T4-encoded polynucleotide kinase
and RNA ligase (7).
The active form of the core polypeptide PrrC has not been
purified to homogeneity. Yet PrrC certainly harbors the ACNase function, judged from the manifestation of ACNase
activity by E. coli (8) and mammalian cells overexpressing
PrrC (9). Moreover, a tRNALys anticodon recognition site has
been pinpointed in a cluster of selected PrrC mutations, two
adjacent members of which (D287Y, S288P) alter the cleavage site specificity (10). Other mutations directed into this
site (D287H, D287Q, or D287N) alter the specificity so that
tRNALys forms with a hypomodified wobble base, otherwise
poor substrates, are rendered more reactive than the natural.
These compensatory effects suggest the existence of a specific
interaction between the Asp287 residue of PrrC and the modified wobble base (11).
Comparing bacterial and mammalian tRNALys substrates of
ACNase has suggested that the specificity determinants reside
in the anticodon loop and lower part of the anticodon stem (9).
These determinants must include the anticodon sequence,
judged from the following observations. First, the anticodon of
tRNALys resembles the anticodons of most secondary substrates cleaved when PrrC is overexpressed (10). Second, most
single anticodon base substitutions in unmodified tRNALys
abolish ACNase reactivity. Third, a substrate analog with a
tRNALys anticodon transplanted in an otherwise tRNAArg sequence is as reactive as the wild type tRNALys sequence (11).
The anticodon stem and loop (ASL) domain of E. coli tRNALys
contains three modified bases that profoundly affect its conformation (12). They include the doubly modified wobble base
5-methylaminomethyl-2-thiouridine (mnm5S2U34), 6-threonyl
carbamoyladenosine 3⬘ to the anticodon (t6A37) and the
pseudouridine of the lower base pair of the stem (⌿39). Comparing the NMR solution structures of a synthetic, fully modified tRNALys ASL and hypomodified counterparts has suggested that mnm5s2U34 and t6A37 rigidify the anticodon in a
predominantly A-RNA stacked conformation. In addition,
t6A37 modestly destabilizes the base pairing interactions of the
ASL while mnm5s2U34 and ⌿39 counteract this effect (12). It is
conceivable that these conformational effects influence ACNase
reactivity. However, some of the modifying groups could also
interact with PrrC directly (11). Although the ASL seems the
prime target of ACNase, weaker interactions with other substrate regions, e.g. the acceptor domain, are not excluded. This
is suggested by the observations that substituting the discriminator base A73 partially inhibits ACNase and truncating the
ACCA 3⬘-overhang relaxes the cleavage site specificity (11).
3836
This paper is available on line at http://www.jbc.org
Substrate Conformation Favored by Anticodon Nuclease
These results indicate that the tRNA acceptor domain helps
position ACNase for the native substrate and that alternative
RNA-protein interactions may form in the ACCA truncated
mutants as well as for A73 mutants.
Here we report that ACNase efficiently cleaves a synthetic
tRNALys ASL containing the base modifications of E. coli
tRNALys. Comparing it with various hypomodified and unmodified analogs revealed opposing effects of the base modifications
on ACNase reactivity. Correlating these data with the observed
contributions of the base modifications to the ASL solution structure (12, 14, 15) leads us to suggest that ACNase prefers substrates where the anticodon nucleotides assume a stacked ARNA conformation and where the base pairing interactions in the
ASL stem are relatively destabilized. We also show that the PrrC
D287H mutation renders human tRNALys3 relatively more reactive than the natural substrate. This finding and the inhibitory
effect of the side chain of the tRNALys-3 modified wobble base
(5-methoxycarbonylmethyl-2-thiouridine, mcm5s2U34) reinforce
the previously proposed interaction between PrrC residue Asp287
and the side chain of the E. coli tRNALys wobble base (11). This
result and the ability to cleave isolated ASLs raise the interesting
prospect of directing ACNase against tRNALys-3 annealed to the
genomic RNA template in the priming complex of the human
immunodeficiency virus.
EXPERIMENTAL PROCEDURES
Materials—All ASL oligonucleotides used in this study are listed in
Table I. The ASL oligonucleotides 1–15 in Table I were chemically
synthesized. The methods have been described for oligonucleotides 1, 3,
4, 6, and 7 (13) oligonucleotides 14 and 15 (16) and the syntheses of
oligonucleotides 8 –11 will be described elsewhere.2 ASL oligonucleotides 16 –23 were transcribed in vitro using T7 RNA polymerase according to an established procedure (17). Those containing inosine (ASLs
20 –22) were transcribed using GMP as primer and ITP instead of GTP.
The in vitro transcribed ASLs were dephosphorylated by calf intestinal
alkaline phosphatase. All ASLs were 5⬘-end labeled using T4 polynucleotide kinase. Bovine tRNALys-3 was a gift from Dr. Roland Marquet,
CNRS Strasbourg. Purified E. coli tRNALys labeled with 32P at phosphate 33p34 was prepared by ligating fragments 1–33 and 34 –76 of
tRNALys purified from T4-infected E. coli prr⫹ cells (7). T7 RNA polymerase and T4 polynucleotide kinase were purchased from U. S. Biochemical Corp., T4 RNA ligase from New England Biolabs, [␥-32P]ATP
from Amersham Biosciences, Inc., poly(U) and trimethylamine-N-oxide
from Sigma. Calf intestinal alkaline phosphatase and Protease Inhibitor Mixture Tablets (Complete Mini) were purchased from Roche Molecular Biochemicals and DNA oligonucleotides from Invitrogen.
Bacteria and Plasmids—E. coli K38:pGP1–2 encoding thermo-inducible T7 RNA polymerase (18) served as host cell for expressing plasmid
pRRC6 (8) and derivatives of plasmid pRRC11 encoding the PrrC mutants D222E (10) or D287H (11).
ACNase Assays—E. coli tRNALys and mammalian tRNALys-3 were
assayed as substrates of wild type and mutant alleles of ACNase using
crude (S-150) fractions essentially as described (11). Assays involving
ASLs required in general the removal of nonspecific nucleases. Therefore, they were performed with a partially purified enzyme fraction
isolated from E. coli K38:pGP1–2:pRRC11-D222E cells by Superdex 200
gel filtration in a procedure to be detailed elsewhere.3 Additional protection against nonspecific degradation was afforded by including in the
reaction mixture carrier poly(U), increasing the trimethylamine-N-oxide concentration to 1.5 M and lowering the reaction temperature from
10 to 0 °C. The standard reaction mixtures (10 ␮l) contained 5 ␮l of the
partially purified core ACNase (D222E allele, Superdex 200 peak activity fraction of ⬃270 kDa) containing 6 ng of PrrC, 36 mM NH4Cl, 6
mM Tris-HCl buffer, pH 7.5, 9 mM MgCl2, 3 mM 2-mercaptoethanol,
50 –100 ng of poly(U), 1.5 M trimethylamine-N-oxide, 6% glycerol, protease inhibitor mixture (Complete, Mini, Roche Molecular Biochemicals, diluted according to manufacturers instructions), 2–3 fmol of 5⬘32
P-labeled ASL or E. coli tRNALys labeled with 32P at the ACNase
cleavage junction. The products were deproteinized and separated by
2
A. C. Bajji and D. R. Davis, manuscript in preparation.
S. Blanga, R. Meidler, M. Amitsur, Y. Jiang, G. Kaufmann, and
A. Azem, manuscript in preparation.
3
3837
denaturing polyacrylamide-urea gel electrophoresis. Each assay was
replicated 3– 4 times. Kinetic analyses were performed with E. coli
tRNALys and several of the ASL substrates found most reactive (listed
in Table II). The kinetic analyses entailed measurements of initial
reaction rates during incubation times reaching 120 min at 1–50 fmol
substrate concentrations. Determinations of cleavage extents were carried out with all the ASL substrates following 3 h incubation. The
amounts of product were determined by counting the radioactivity or by
densitometric tracing of the gel autoradiogram using Hewlett Packard
ScanJet 3p and TINA software (Raytest Isotopenmessgeräte GmbH),
compatible with the TINA-PCBAS and TIFF files of the scanner.
RESULTS
Specific and Efficient Cleavage of the Fully Modified tRNALys
ASL by ACNase—An ASL 17-mer containing the three modified bases of E. coli tRNALys (henceforth the reference ASL,
Fig. 1), various hypomodified and unmodified derivatives and
ASLs containing modified bases of mammalian tRNALys3 (Table I) were examined as ACNase substrates. The reference ASL
and other chemically synthesized ASLs (ASLs 1–15) matched
the mammalian tRNALys-3 sequence, which differs from that of
E. coli tRNALys in the upper three base pairs of the anticodon
stem (compare the left and right panels in Fig. 1). However, the
upper portion of the stem was considered nonessential for
recognition by ACNase (9). The synthetic ASLs 5, 12, and 13
contained an extra 3⬘-dT residue since a labile linker was
utilized for the chemical synthesis of the ASLs containing
mcm5s2U and ms2t6A.2 The unmodified ASL 15 featuring the
mammalian tRNALys3 sequence was also chemically synthesized. Unmodified ASLs 16 –23 were transcribed in vitro and all
of them contained a 5⬘ terminal G residue dictated by the
constraints of transcription with T7 RNA polymerase (17). The
in vitro transcribed ASL 16 corresponded in sequence to E. coli
tRNALys (Fig. 1b). ASL 19 resembled the E. coli tRNAVal-3 ASL
sequence (19) except for a reversed 5⬘ to 3⬘ terminal GC base
pair. The DNA templates of ASL 19 and of two other ASLs with
arbitrary GC-rich stem sequences (ASLs 17 and 18) were also
transcribed into inosine-rich versions (ASLs 20 –22). ASL 23
was a mutant of E. coli tRNALys sequence in which A37 was
replaced by G.
Incubation of the 5⬘-32P-labeled reference ASL with a partially purified ACNase preparation under conditions that minimize nonspecific degradation (“Experimental Procedures”)
yielded a heptanucleotide-like product missing from the
ACNase-negative control (Fig. 2, compare lanes 2 and 3 with
lane 4). This product had a slightly reduced electrophoretic
mobility compared with a heptanucleotide containing a (2⬘)
3⬘-terminal phosphate within a marker ladder generated by
partial alkaline hydrolysis of the reference ASL (Fig. 2, compare lanes 1 and 4). This retardation of the ACNase product
could be accounted for by the lower negative charge of the
2⬘:3⬘-cyclic phosphate end-group expected to be found in the
product. Incubating the ACNase product with T4 polynucleotide kinase, endowed with 2⬘:3⬘-cyclic-phosphodiesterase and
3⬘-phosphatase activities (7, 20), shifted the product to the
position of the 3⬘-dephosphorylated heptanucleotide marker
(compare lanes 5, 6), consistent with removal of the cyclic
phosphate end group. Hence, ACNase appeared to cleave the
ASL similar to the natural substrate, targeting the same site
and producing the same cleavage termini.
Kinetic comparison of the reference ASL and full sized
tRNALys (Table II) yielded respective Kcat (for a PrrC-D222E
hexamer) of ⬃0.04 min⫺1 and ⬃0.02 min⫺1 and Km of 2.6 ⫻
10⫺7 M and 0.8 ⫻ 10⫺7 M. A 2-fold faster cleavage of the ASL
was also demonstrated by digesting the two substrates in the
same reaction mixture (Fig. 3).
Contributions of ASL Base Modifications to ACNase Reactivity—In vitro transcribed, unmodified tRNALys is less reactive
3838
Substrate Conformation Favored by Anticodon Nuclease
FIG. 1. Composition of the tRNA ASLs. The sequence of the reference ASL is derived from mammalian tRNALys-3, which differs from E. coli
tRNALys in the upper three bases of the anticodon stem. The mammalian tRNALys-3 isoacceptor contains the modified wobble base 5-methoxycarbonylmethyl-2-thiouridine (mcm5s2U or U9), the modified base 3⬘ to the anticodon is 2-methylthio-6-threonylcarbamoyl-adenosine (ms2t6A or
A9), and the C-nucleoside pseudouridine (⌿) stabilizes the A31-⌿39 base pair. The single E. coli tRNALys isoacceptor has a related modification
pattern with 5-methylaminomethyl-2-thiouridine (mmm5s2U or U8), 6-threonylcarbamoyl-adenosine (t6A or A7), and pseudouridine (⌿). The
modified nucleoside abbreviations are those originally described by Sprinzl et al. (29). The updated nucleoside modification data base is maintained
by McCloskey and co-workers (30).
TABLE I
Extents of cleavage of ASL substrates by ACNase
ASL
Stema
Modified bases and extra dT
% Cleavageb
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
Lys3
Lys3
Lys3
Lys3
Lys3
Lys3
Lys3
Lys3
Lys3
Lys3
Lys3
Lys3
Lys3
Lys3
Lys3
EC
GC-rich 1
GC-rich 2
Val3-like (G)
IC-rich 1
IC-rich 2
Val3-like (I)
EC Ala37 3 Gly
mnm2s2U34/t6A37/⌿39
ms2t6A37/⌿39
t6A37/⌿39
t6A37
mcm5s2U34/ms2t6A37/⌿39/3⬘-dT
s2U34/⌿39
mnm5s2U34/⌿39
mcm5s2U34
mcm5s2U34/⌿39
mcm5U34
mcm5U34/⌿39
ms2t6A37/3⬘dT
ms2t6A37/⌿39/3⬘-T
⌿39
60 ⫾ 3
73 ⫾ 4
70 ⫾ 2
73 ⫾ 5
8⫾1
2 ⫾ 0.3
7.2 ⫾ 0.8
1.1 ⫾ 0.2
0.2 ⫾ 0.1
1.1 ⫾ 0.3
0.2 ⫾ 0.1
43 ⫾ 2
30 ⫾ 3
1.8 ⫾ 0.3
3.8 ⫾ 0.4
4 ⫾ 0.5
NDc
ND
ND
3 ⫾ 0.5
2 ⫾ 0.3
0.4 ⫾ 0.1
4 ⫾ 0.6
a
The unmodified Lys3 ASL sequence is 5⬘-UCAGACUUUUAAUCUGA-3⬘. The unmodified E. coli ASL sequence is pGUUGACUUUUAAUCAAC. The arbitrary GC-rich ASLs 1 and 2 are pGUUGACUUUUAAUCAAC and pGGCGGCUUUUAACCGCC, respectively. The Val3-like
ASL resembles that of E. coli tRNAVal3 except for reversal of the 5⬘ to 3⬘
base pair. The IC-rich counterparts of the GC-rich and Val3-like ASL
contain I instead of G except for the 5⬘ most G. EC A37 3 G is a mutant
of the unmodified ASL corresponding to E. coli tRNALys.
b
Extents of cleavage were measured in standard reaction mixtures
incubated for 3 h.
c
ND, not detectable.
than the natural, fully modified ACNase substrate, suggesting
a role for at least some of the base modification in substrate
recognition and/or reactivity. The tRNALys base modifications
relevant to ACNase reactivity could be confined to the ASL
domain, judged by the high reactivity of the reference ASL (Fig.
2, Table II). The importance of at least one of the wobble base
modifications to ACNase reactivity has been previously suggested by inefficient cleavage of hypomodified tRNALys lacking
the mnm5U34 modification (both in vivo and in vitro) or s2U34
(in vivo) and suppression of the wobble base lesions by certain
PrrC Asp287 replacement mutations (11). To investigate the
contribution of all the ASL base modifications to ACNase reactivity under more defined conditions, we compared the reference ASL with the analogs listed in Table I in an in vitro
ACNase assay. Apparent Km and Kcat values were obtained for
the most reactive ASLs (Table II). Weaker substrates were
characterized by extents of cleavage under standard assay
conditions (Table I). Below we use these data to assess the
contributions of individual base modifications to ACNase reactivity, comparing matched ASL pairs differing by the presence
of a given base modification.
The most pronounced effect was exerted by the bacterial
t6A37 and mammalian ms2t6A37 modifications. Hypomodified
ASLs containing either as the only modified base were cleaved
to a 17–18-fold greater extent than the corresponding unmodified ASL. These two partially modified ASLs resembled the
reference ASL in Km but had at least 2-fold larger Kcat values
(Table II). Hence, the mnm5s2U34 and/or ⌿39 base modifications could attenuate ACNase reactivity of the reference ASL.
The specific contributions of the wobble base modifications to
ACNase reactivity were evaluated similarly. The extent of
cleavage of the mnm5s2U34/⌿39-ASL was 3– 4-fold larger than
that of the s2U34/⌿39-ASL (Table I). This suggested that the
mnm5U34 side chain exerts a positive effect on ACNase reactivity. In contrast, the mcm5U34-ASL and mcm5s2U34-ASL,
both containing the side chain of the tRNALys-3 wobble base,
were less reactive than the corresponding unmodified ASL
(Table I). The opposing effects of mnm5U34 and mcm5U34 were
underscored by the large difference in the extents of cleavage
between the mnm5s2U34/⌿39 and mcm5s2U34/⌿39 containing
ASLs (7.2 versus less than 0.2%, respectively). The negative
effect of the mcm5U34 was further demonstrated by the different kinetic values of the ms2t6A37/⌿39/3⬘-dT ASL (Km ⫽ 3.0 ⫾
0.6 ⫻ 10⫺8 M, Kcat ⫽ 0.014 min⫺1) and mcm5s2U34/ms2t6A37/
Substrate Conformation Favored by Anticodon Nuclease
FIG. 2. ACNase specifically cleaves the reference ASL. The 5⬘P-labeled reference ASL was incubated with partially purified ACNase (Superdex 200 fraction) and the products were separated by
denaturing 20% polyacrylamide-urea gel electrophoresis as such (lane
4) or after treatment with T4 polynucleotide kinase (lane 5). A marker
“ladder” (lane 1) was obtained by partial alkaline hydrolysis of the ASL.
Part of the digest was further treated with polynucleotide kinase that
effectively dephosphorylates tri- and higher oligonucleotides (lane 6).
The gap between the heptamer and octamer markers in lanes 1 and 6 is
due to the presence of the basic mnm5s2U34 residue in the octamer.
Numbers on the right are chain length in nucleotides; numbers on the
left marked by an asterisk represent the slower migrating 3⬘-OH containing oligonucleotides. Lanes 2 and 3 are respective controls of untreated ASL or ASL incubated with inactivated ACNase (Superdex
fraction preincubated 30 min at 37 °C).
3839
32
TABLE II
Kinetic parameters of different ASL substrates of ACNase
Substrate
Kcata
min
E. coli tRNALys
mnm5s2U34/t6A37/⌿39-ASL
ms2t6A37/⌿39-ASL
t6A37/⌿39-ASL
t6A37-ASL
ms2t6A37/3⬘-dT-ASL
ms2t6A37/⌿39/3⬘-dT-ASL
mcm5s2U34/ms2t6A37/⌿39/3⬘-dT-ASL
a
⫺1
0.018 ⫾ 0.005
0.044 ⫾ 0.009
0.199 ⫾ 0.091
0.145 ⫾ 0.055
0.109 ⫾ 0.073
0.016 ⫾ 0.004
0.014 ⫾ 0.0005
0.004 ⫾ 0.0005
KM
M
10⫺8
8⫾2
26 ⫾ 6
30 ⫾ 10
17 ⫾ 7
60 ⫾ 30
3.8 ⫾ 0.8
3.0 ⫾ 0.6
6.0 ⫾ 2.0
Reaction mixtures (20 ␮l) contained 12 ng of PrrC (2.2 nM hexamer).
⌿39/3⬘-dT ASL (Km ⫽ 6.0 ⫾ 2.0 ⫻ 10⫺8 M, Kcat ⫽ 0.004 min⫺1).
Thus, mcm5s2U34 reduced the Kcat by more than 3-fold and the
binding affinity by ⬃2-fold and the overall catalytic efficiency
⬃7-fold. On the other hand, adding mnm5s2U34 to the t6A/⌿39
background increased Kcat by more than 3-fold and reduced the
affinity ⬃2-fold (Km ⫽ 2.6 ⫾ 0.6 ⫻ 10⫺7 M, Kcat ⫽ 0.044 min⫺1
versus Km ⫽ 1.7 ⫾ 0.7 ⫻ 10⫺7 M, Kcat ⫽ 0.144 min⫺1, respectively). Hence, the negative changes in catalytic efficiency con-
FIG. 3. E. coli tRNALys and the reference ASL as ACNase substrates. The two substrates were incubated in the same reaction mixture. RNA extracted from aliquots withdrawn at the indicated times
was separated by denaturing polyacrylamide-urea gel electrophoresis.
ASL frag, heptanucleotide cleavage product of the ASL.
ferred by mcm5s2U34 may be ascribed to the side chain rather
than 2-thiol group. This inference is supported by the similar
inhibitions caused by mcm5U34 and mcm5s2U34 when introduced into the unmodified background (Table I, compare ASLs
8 and 10 with 15). It is noteworthy that the lack of the s2U34
modification from E. coli tRNALys had no detectable effect on
the cleavage efficiency in vitro although in vivo this lesion was
synthetically lethal with PrrC and severely inhibited tRNALys
cleavage (11).
The fully modified E. coli tRNALys mnm5s2U34/t6A37/⌿39ASL was cleaved with a 10-fold higher Kcat and exhibited a
3– 4-fold weaker binding compared with mcm5s2U34/ms2t6A37/
⌿39/3⬘-dT-ASL carrying the base modifications of tRNALys-3
(Table II). However, confounding this comparison is the large
negative effect of the extra 3⬘-dT residue in the latter substrate.
Namely, against the ms2t6A37/⌿39 backdrop, the extra 3⬘-dT
reduced the Km and Kcat each by about an order of magnitude
(Table II).
The presence of ⌿39 instead of U39 rendered the ASL in
certain cases less reactive. The unmodified ASL was cleaved to
a 2-fold higher extent than the ⌿39-containing counterpart.
Similarly, introducing ⌿39 into ASLs containing mcm5U34 or
3840
Substrate Conformation Favored by Anticodon Nuclease
FIG. 4. Mutation D287H renders tRNALys-3 more reactive than
E. coli tRNALys. The two tRNA substrates were incubated in the
standard reaction mixture containing the indicated PrrC alleles. The
products were separated by denaturing polyacrylamide urea gel electrophoresis as described in the legend to Fig. 3. The ratios between the
reactivities of the two substrates were calculated from the initial rates
of cleavage. EC, E. coli tRNALys; Lys3, tRNALys-3.
mcm5s2U34 reduced their activity more than 5-fold (Table I).
However, ⌿39 had no apparent effect on either kinetic parameter
when introduced in the ms2t6A37/3⬘-dT context and over the
t6A37 background it even reduced the Km ⬍3-fold (Table II).
ASL Reactivity and Stem Stability—Unmodified ASLs with
stems (underlined) corresponding in sequence to mammalian
tRNALys3 (5⬘-UCAGACUUUUAAUCUGA-3⬘) or E. coli tRNALys
(5⬘-GUUGACUUUUAAUCAAC-3⬘) were similarly reactive as
ACNase substrates, confirming the assumption that the top
three base pairs are not critical for ACNase specificity (9).
Arbitrary, all GC stems of the respective ASLs 17 and 18
(5⬘-GGCGGCUUUUAACCGCC-3⬘ and 5⬘-GCCGGCUUUUAAGCGGC-3⬘) abolished the reactivity. A similar effect was
exerted by the GC-rich stem of ASL 19, resembling that of E.
coli tRNAVal-3 except for a reversed 5⬘ to 3⬘ terminal pair
(5⬘-GCACCCUUUUAAGGUGC-3⬘). Replacing the G residues
of these ASLs with inosine residues (except for the 5⬘-terminal)
restored the reactivity almost to the level of the unmodified
reference ASLs in the case of the two arbitrary stem sequences.
However, the tRNAVal-3-like I-rich ASL was an order of magnitude less reactive.
A PrrC Mutation Renders tRNALys-3 More Reactive Than the
Natural Substrate—The D287H, D287N, or D287Q mutations
have rendered hypomodified E. coli tRNALys forms containing
s2U34 or mnm5U34 more reactive than the natural substrate,
opposite to the preference of wild type PrrC (11). These effects,
attributed to some interaction of Asp287 with the wobble base,
have also raised the prospect of designing new ACNase cleavage specificities (10), especially for a differently modified wobble base such as that of mammalian tRNALys-3. In fact, whereas
wild type PrrC or the pseudo-wild type allele D222E (11)
cleaved E. coli tRNALys 2–3 faster than mammalian tRNALys-3,
D287H featured the opposite preference (Fig. 4).
DISCUSSION
The tRNALys ASL as a Minimal Essential Substrate of
ACNase—The reference ASL with the three modified bases of
E. coli tRNALys was cleaved by ACNase twice as fast as the
natural substrate but featured a 3– 4-fold reduced affinity to
the enzyme, suggesting that the ASL domain contains all or
nearly all of the ACNase recognition determinants. The small
differences between the ASL and full-sized tRNA in Kcat and
Km are attributed to greater conformational freedom of the
ASL. Such flexibility could facilitate attainment of a productive
transition-state conformation by the ASL but weaken initial
binding to the enzyme. However, the weaker binding of the
ASL also leaves open possible interactions between PrrC and
other portions of tRNALys such as the acceptor region. Namely,
mutating the A73 discriminator base inhibits ACNase and
truncating the 3⬘-terminal ACCA sequence relaxes the cleavage site specificity (11). These outcomes could be explained by
a weak interaction between PrrC and the acceptor region of
tRNALys. Accordingly, the A73 discriminator could contribute
to substrate specificity, directly or by counter-selecting other
tRNAs. We also speculate that truncation of the substrate to
contain just the ASL eliminates these interactions that cause
misalignment and therefore allows for specific and efficient
recognition of the cleavage site.
Structural ASL Features Favored by ACNase—Comparing
the NMR solution structures of the reference ASL and hypomodified counterparts has suggested that individual base modifications play distinct and partially opposing roles in transforming the disordered, unmodified tRNALys ASL into the
highly ordered native structure (12). Thus, t6A37 improves
stacking with A38, and strengthens the interaction of U33 N3H
with the 35p36 phosphate, a hallmark of the anticodon loop
U-turn. On the other hand, t6A37 partially destabilizes the
ASL stem base pairing, reducing the Tm by about 2 °C. The
wobble base side chain mnm5U34 also stabilizes the anticodon
base stacking and U-turn conformation but partially counteracts the base pair disrupting effect of t6A37. Destabilization of
the stem and/or the C32-A38 base pair (21) by t6A37 also opposes the effect of the ⌿39 modification (12, 14, 15). Here we
have shown that the ASL base modifications also exert distinct
effects on ACNase reactivity (Tables I and II). The underlying
causes of these complicated and opposing effects may be understood by considering the contributions of the individual base
modifications to the ASL solution structure, as described
above. Such a correlation leads us to propose that ACNase
favors substrates where the anticodon nucleotides are held in a
helical A-RNA conformation and the U-turn conformation is
retained but the ASL stem base pairing interactions are modestly destabilized. This assumption is backed by the following
arguments. First, t6A37 or ms2t6A37 that dramatically enhanced the ACNase reaction rate also stabilize the anticodon
A-RNA and the loops U-turn conformations and destabilize the
anticodon stem and bifurcated C32-A38 base pair (12, 14), suggesting that ACNase favors these structural features. In agreement, ⌿39, which inhibited ACNase activity in certain cases,
exerts the opposite effect on ASL base pairing interactions. As
for the mild positive effect of mnm5U34 on ACNase reactivity,
it could reflect a balance between a positive contribution to
ACNase reactivity of the enhanced anticodon stacking by this
modification and a negative contribution due to the stabilized
base pairing. However, part of the ACNase-stimulating effect
of mnm5U34 may be attributed to a direct interaction with
Asp287, the anticodon-recognizing residue of PrrC (an issue to
be elaborated below). Abolition of ACNase reactivity by the
GC-rich stems, but not the IC-rich, reinforces the notion that
base pairing stability of the ASL negatively affects the reactivity. As already mentioned, the higher Kcat and lower Km of the
reference ASL compared with the full-sized tRNALys may be
attributed to the fact that the upper base pairs of the stem are
more constrained within the intact tRNA structure. Hence, the
destabilization of the anticodon stem, needed for the productive
interaction with ACNase, may be inhibited with intact
tRNALys. However, as we have seen for the ASLs, the more
rigid conformation of the intact tRNA could promote tighter
initial binding to the enzyme.
Substrate Conformation Favored by Anticodon Nuclease
Transplanting the tRNALys UUU anticodon within an
otherwise tRNAVal-3(UUU) sequence does not elicit detectable
ACNase reactivity whereas tRNAArg(UUU) is highly reactive
(11). The difference between the two chimerical tRNA sequences has been attributed to ill-defined ACNase determinants found outside the anticodon loop and shared by tRNALys
and tRNAArg but missing from tRNAVal-3. However, in view of
the current data, a new interpretation becomes apparent.
Namely, the failure to cleave tRNAVal-3(UUU) may reflect the
greater stability of its GC-rich anticodon stem (4 GC pairs) and
the reversed purine-pyrimidine configuration of the two base
pairs at the bottom of the stem. The importance of this configuration is suggested by the weaker reactivity of the IC-rich
version of the tRNAVal-3-ASL, compared with the counterparts
with arbitrary stem sequences that shared the tRNALys/
tRNAArg configuration. Presumably, the reversed base pair
configuration further stabilizes the stem by enhancing G39-A38
base stacking.
A Distinct Wobble Base Side Chain Causes the Reduced Reactivity of tRNALys-3—The relative ACNase reactivities of natural substrate, the completely unmodified version and mutants
thereof as well as of the mammalian tRNALys isoacceptors have
suggested the following order of wobble base preference of
ACNase: mnm5s2U⬎U⬎C⬎mcm5s2U (11). However, such an
order has been derived from eclectic data collected in vivo and
in vitro and comparisons of substrates differing in more that
just their wobble bases. The current comparison uses defined in
vitro assay conditions of matched ASLs differing only in a
wobble base modification (Tables I and II). These results confirm the opposing contributions of the bacterial mnm5U34 and
mammalian mcm5U34 side chains to ACNase reactivity,
whereas both modifications similarly influence the overall ASL
conformation (12, 24). Therefore, the inhibitory effect of
mcm5U34 on ACNase reactivity suggests a specific interaction
between residue Asp287 of PrrC and the mnm5U34 side chain of
the natural substrate (11). Since replacing mnm5U34 with
mcm5U34 reduces both Km and Kcat (Table II), the Asp287mnm5U34 interaction could influence both substrate binding
and the catalytic step.
The detection of PrrC mutations that alter the substrate
cleavage specificity and compensate for a missing wobble base
modification (11) raised the possibility that some of these mutations would also cause ACNase to prefer mammalian
tRNALys-3 over the natural substrate. One interest in such an
outcome stems from the role human tRNALys-3 plays as the
primer tRNA of reverse transcription in human immunodeficiency viruses (22, 23). There are several characteristics of the
tRNALys-3 primer that raise the prospect of using ACNase as a
model for developing anti-HIV therapeutics. The tRNALys-3
primer is indifferent to viral genetic drift (25) and the tRNA
anticodon interaction with the HIV A-loop is critical for transcription initiation (26). Provided that the tRNA anticodon is
accessible to ACNase in the primer-template complex, it may
be possible to tailor ACNase to discriminate between the free
form of tRNALys-3 and the annealed tRNA. Detection of
ACNase derivatives more proficient in cleaving tRNALys-3
would constitute a step toward the goal of developing a model
system for therapeutics targeted at the primer-template complex. Since the reduced reactivity of tRNALys-3 is likely due to
the presence of mcm5U34 instead of mnm5U34 in the natural
substrate, it was expected that tRNALys-3 will be rendered
3841
more reactive by some of the PrrC mutations that compensate
for the absence of mnm5U34 (11). This expectation was confirmed by the in vitro behavior of the D287H allele, which
cleaved tRNALys-3 relatively faster than wild type and pseudowild type alleles, whereas the latter two alleles were relatively
more reactive with E. coli tRNALys (Fig. 4). Mechanistically,
the reversal of substrate preference may be explained in that
the mnm5U34-Asp287 and mcm5U34-D287H pairs form saltbridges or hydrogen bond interactions of opposite polarities
that are vital for substrate binding and/or reactivity. Interestingly, among the PrrC D287 replacement mutants expressed in
mammalian cells D287Q showed the highest activity with
tRNALys-3, cleaving it to a severalfold higher extent than wild
type PrrC.4 The efficient cleavage of the isolated ASL domain
by ACNase also suggests that partially melted tRNALys-3 annealed to the HIV-1 genomic RNA will be recognized by
ACNase since the native ASL conformation would be retained
in the primer-template complex (27, 28). This expectation has
been recently confirmed.5
REFERENCES
1. Kaufmann, G. (2000) Trends Biochem. Sci. 25, 70 –74
2. Levitz, R., Chapman, D., Amitsur, M., Green, R., Snyder, L., and Kaufmann,
G. (1990) EMBO J. 9, 1383–1389
3. Linder, P., Doelz, R., Gubler, M., and Bickle, T. A. (1990) Nucleic Acids Res. 18,
7170
4. Amitsur, M., Morad, I., Chapman-Shimshoni, D., and Kaufmann, G. (1992)
EMBO J. 11, 3129 –3134
5. Tyndall, C., Meister, J., and Bickle, T. A. (1994) J. Mol. Biol. 237, 266 –274
6. Penner, M., Morad, I., Snyder, L., and Kaufmann, G. (1995) J. Mol. Biol. 249,
857– 868
7. Amitsur, M., Levitz, R., and Kaufmann, G. (1987) EMBO J. 6, 2499 –2503
8. Morad, I., Chapman-Shimshoni, D., Amitsur, M., and Kaufmann, G. (1993)
J. Biol. Chem. 268, 26842–26849
9. Shterman, N., Elroy-Stein, O., Morad, I., Amitsur, M., and Kaufmann, G.
(1995) Nucleic Acids Res. 23, 1744 –1749
10. Meidler, R., Morad, I., Amitsur, M., Inokuchi, H., and Kaufmann, G. (1999) J.
Mol. Biol. 287, 499 –510
11. Jiang, Y., Meidler, R., Amitsur, M., and Kaufmann, G. (2001) J. Mol. Biol. 305,
377–388
12. Sundaram, M., Durant, P. C., and Davis, D. R. (2000) Biochemistry 39,
12575–12584
13. Sundaram, M., Crain, P. F., and Davis, D. R. (2000) J. Org. Chem. 65,
5609 –5614
14. Stuart, J. W., Gdaniec, Z., Guenther, R., Marszalek, M., Sochacka, E.,
Malkiewicz, A., and Agris, P. F. (2000) Biochemistry 39, 13396 –13404
15. Durant, P. C., and Davis, D. R. (1999) J. Mol. Biol. 285, 115–131
16. Bajji, A. C., and Davis, D. R. (2000) Org. Lett. 2, 3865–3868
17. Milligan, J. F., and Uhlenbeck, O. C. (1989) Methods Enzymol. 180, 51– 62
18. Tabor, S., and Richardson, C. C. (1985) Proc. Natl. Acad. Sci. U. S. A. 82,
1074 –1078
19. Sprinzl, M., Horn, C., Brown, M., Ioudovitch, A., and Steinberg, S. (1998)
Nucleic Acids Res. 26, 148 –153
20. Cameron, V., and Uhlenbeck, O. C. (1977) Biochemistry 16, 5120 –5126
21. Auffinger, P., and Westhof, E. (1999) J. Mol. Biol. 292, 467– 483
22. Mak, J., and Kleiman, L. (1997) J. Virol. 71, 8087– 8095
23. Arts, E. J., Miller, J. T., Ehresmann, B., and Le Grice, S. F. (1998) J. Biol.
Chem. 273, 14523–14532
24. Benas, P., Bec, G., Keith, G., Marquet, R., Ehresmann, C., Ehresmann, B., and
Dumas, P. (2000) RNA 6, 1347–1355
25. Wakefield, J. K., Wolf, A. G., and Morrow, C. D. (1995) J. Virol. 69, 6021– 6029
26. Huang, Y., Shalom, A., Li, A., Wang, J., Mak, J., Wainberg, M. A., and
Kleiman, L. (1996) J. Virol. 70, 4700 – 4706
27. Puglisi, E. V., and Puglisi, J. D. (1998) Nat. Struct. Biol. 5, 1033–1036
28. Elgavish, T., VanLoock, M. S., and Harvey, S. C. (1999) J. Mol. Biol. 285,
449 – 453
29. Sprinzl, M., Hartmann, T., Weber, J., Blank, J., and Zeidler, R. (1989) Nucleic
Acids Res. 17, r1–r172
30. Rozenski, J., Crain, P. F., and McCloskey, J. A. (1999) Nucleic Acids Res. 27,
196 –197
4
M. Perlman, M. Amitsur, O. Elroi-Stein, L. Kleiman, and G.
Kaufmann, manuscript in preparation.
5
M. Perlman, C. Isel, R. Marquet, and G. Kaufmann, unpublished
results.